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S Leenstra

Publications and source records attributed to S Leenstra.

15 recordsLinked to original sources
PubMed1998

Genetic sub-types of human malignant astrocytoma correlate with survival.

In human malignant astrocytoma, age of the patient and histological grade of the tumor are important prognostic variables. Several genetic changes have been reported to occur in these tumors, which may be of additional and independent prognostic relevance. To determine their prognostic significance, we analyzed 75 high-grade tumors, 12 anaplastic astrocytomas and 63 glioblastomas multiforme, for the presence of genetic changes that occur frequently in high-grade astrocytoma, i.e., loss of heterozygosity (LOH) for chromosome 10, p53-gene alteration (mutation and/or LOH), and EGFR-gene amplification. We defined 4 groups of patients who showed a specific combination of genetic changes in the tumor: group 1, p53-gene alteration without complete LOH 10; group 2, complete LOH 10 only; group 3, p53-gene alteration + complete LOH 10; group 4, complete LOH 10 + EGFR-gene amplification. In univariate analysis, the log-rank test revealed significant differences in survival between patients of group 1 (median survival of 13 months) and group 3 (median survival of 5.2 months, p = 0.0058) and between patients of group 1 and group 4 (median survival of 4 months, p = 0.0033). In multivariate analysis, age and genetic sub-type proved to be important prognostic variables, whereas histological grading was less important. The age-corrected survival time for group-4 patients is significantly shorter than that for group-1 patients (relative risk = 3.79, p = 0.0075). Our data indicate that genetic sub-type is an important prognostic variable in human high-grade astrocytoma.

Adult↗
PubMed1998

Dynamics of genetic alterations associated with glioma recurrence.

We investigated the dynamics of the genetic changes that are associated with two types of glioma recurrence, that is, progression from a lower-grade to a high-grade tumor (7 cases) and development of a same high-grade recurrence (15 cases). Each pair of tumors was analyzed for TP53 mutation, EGFR amplification, and loss of heterozygosity for tumor suppressor genes (TP53, RB1, CDKN2A, PTEN, DMBT1) and tumor suppressor gene regions (1p36, 19q13, 11p15, 10p15) known to be frequently implicated in glioma tumorigenesis. By comparing the genetic changes in the primary and corresponding secondary tumors, we found that additional loss of CDKN2A and/or RB1, encoding important components of the cell cycle regulatory pathway, was the most frequent genetic change in both types of recurrence development (10 of 22 cases, 45%). Additional loss of heterozygosity for the 10p15 region, for PTEN, and/or for DMBT1 in the recurrent tumor was noted in 7 of 22 cases (32%), suggesting that additional inactivation of tumor suppressor genes on chromosome 10 is another important feature of glioma relapse. Less frequent additional losses were detected for chromosome regions 11p15 and 19q13 (3 of 22 cases, 14%, each). We conclude that glioma recurrences are characterized by an increased involvement of tumor suppressor genes, even in those cases in which the primary and secondary tumor are of the same high malignancy grade.

Adult↗
PubMed1997

Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas.

The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the radiation effect of glioblastomas. The advantage of OMS is maintenance of the characteristics of the original tumour, which is lost in conventional cell cultures. OMS prepared from four glioblastomas were treated with hypofractionated radiation with a radiobiologically equivalent dose to standard radiation treatment for glioblastoma patients. After treatment, the histology as well as the cell proliferation of the OMS was examined. After radiation, a significant decrease in cell proliferation was found, although no histological damage to the OMS was observed. The modest effects of radiation on the OMS are in agreement with the limited therapeutic value of radiotherapy for glioblastoma patients. Therefore, OMS seems to be a good alternative for cell lines to study the radiobiological effect on glioblastomas.

Cell Division↗
PubMed1997

Malignant astrocytoma-derived region of common amplification in chromosomal band 17p12 is frequently amplified in high-grade osteosarcomas.

Recently, we reported a new amplification event that involves marker D17S67 in 17p12 in three malignant astrocytomas of patients with a very short survival. The amplified region may contain an oncogene implicated in astrocytoma tumorigenesis. To determine the extent of the amplified regions, we constructed a yeast artificial chromosome contig spanning the D17S67 region and tested the amplification status of markers that map to the contig. We determined a commonly amplified region between markers D17S1311 and D17S1875 with a maximal length of 1,630 kb. By using marker 745R, from within the commonly amplified region, we screened 60 high-grade astrocytomas but could not detect additional tumors with the amplification event. This suggests that the incidence of the amplification event in high-grade astrocytoma is low (5%). It has recently been shown by comparative genomic hybridization that amplification of 17p11-p12 is a frequent event in high-grade osteosarcomas, occurring in 20-30% of cases. Since the commonly amplified region is within 17p12, we tested 745R in 20 osteosarcomas, including 6 lung metastases, and detected amplification in 9 cases (45%). Marker 745R was found to be amplified in 4 of the 6 lung metastases (66%). From this frequent involvement and the association with clinically aggressive astrocytomas we conclude that for both tumor types presence of the amplification event seems to correlate with aggressive clinical behaviour.

Astrocytoma↗
PubMed1996

Daunorubicin and doxorubicin but not BCNU have deleterious effects on organotypic multicellular spheroids of gliomas.

In the present study organotypic multicellular spheroids (OMS) were used to study the effects of chemotherapeutic agents on malignant gliomas. Compared with the frequently used cell line models, OMS have several advantages with respect to the preservation of the cellular heterogeneity and the structure of the original tumour. OMS prepared from seven glioma specimens were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), daunorubicin or doxorubicin. After exposure to these drugs, the histology and cell proliferation of the OMS were analysed by immunohistochemistry and image analysis. Furthermore, the expression of P-glycoprotein (P-gp) and multidrug resistance-related protein (MRP), which both can contribute to resistance to daunorubicin and doxorubicin, were immunohistochemically investigated. We found that OMS from gliomas are sensitive for daunorubicin and doxorubicin but not for BCNU in terms of tissue destruction and decrease in cell proliferation. In addition, all gliomas were P-gp and MRP negative, which is in accordance with the sensitivity for daunorubicin and doxorubicin. Considering the potential use of several new alternative drug delivery methods, such as intratumoural implantation of drug-impregnated polymers or liposomal encapsulation of cytostatic drugs, daunorubicin and doxorubicin might be effective in the treatment of malignant gliomas.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Expression of CD44 splice variants in human primary brain tumors.

Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of CD44 variant exon encoded sequences (CD44v) in primary brain tumors in situ has not been studied in detail. In the present study, the expression of CD44s and CD44v epitopes was analyzed immunohistochemically on frozen sections of primary brain tumors. In addition, the expression of CD44 on cultured glioma cells was investigated by immunofluorescence flow cytometry. The results demonstrate the presence of CD44s epitopes and of CD44 splice variants containing CD44v4, v5 and v10 sequences in various types of brain tumors. A subgroup of highly malignant gliomas showed a strong (focal) expression of CD44v5. CD44v6 was absent in all brain tumors examined. CD44s appeared to be the dominant form of CD44 expressed in primary brain tumors, its expression was not correlated with tumor grade. We envisage that CD44 isoforms, in particular CD44s, may contribute to the invasive character of primary tumors by interacting with hyaluronate, one of the most abundant molecules in the extracellular matrix of the brain.

Alternative Splicing↗
PubMed1995

Human malignant astrocytes express macrophage phenotype.

Six well-characterized specimens of cultured astrocytoma cells were investigated with a panel of macrophage markers. Our results show that the macrophage markers OKM-1(CD11b), OKM5(CD36), EBM11(CD68), HAM56, Factor 13, alpha-1-antichymotrypsin, alpha-1-antitrypsin, ferritin and lysozyme are clearly reactive to neoplastic astrocytes whereas astrocytes in normal brain specimens are not reactive. In order to obtain further confirmation concerning the reactivity of tumor cells in vivo, we simultaneously measured by flow cytometric analysis DNA content and HAM56 immunoreactivity in a freshly obtained tumor specimen. In this experiment we found a marked reactivity of aneuploid cells to HAM56. The macrophage phenotype of malignant astrocytes may reflect a similarity in functions of these cells and tumor-associated macrophages which promote tumor growth via the production of growth factors and angiogenic factors. Furthermore, our findings implicate that demonstration of macrophages within malignant astrocytomas by using macrophage-specific antibodies must be cautiously considered.

Astrocytes↗
PubMed1995

Long-term culture of organotypic multicellular glioma spheroids: a good culture model for studying gliomas.

Gliomas, as well as other solid tumours, contain tumour stroma composed of connective tissue, macrophages, capillaries and other non-cellular constituents. Therefore, a homogeneous culture of tumour cells alone, as is often used as a culture model for gliomas, is not ideal to study all aspects of gliomas. In the present study we describe an alternative culture model, i.e. organotypic multicellular spheroids (OMS), that histologically closely resembles the tumour in vivo. Glioma explants, obtained at surgery from five patients, were cultured on agarose to form OMS, which were cultured for up to 16 weeks. At regular intervals, OMS were fixed and histological and immunocytochemical analyses were carried out. The histology as well as the immunocytochemical characteristics of the OMS proved to be almost unchanged after a culture period of 16 weeks. In contrast to monolayer cultures, glial fibrillary acidic protein (GFAP) expression in the OMS is preserved after 16 weeks of culture. However, in OMS from three out of five patients, small GFAP-negative cells appeared in the outer cell layers between 1 and 2 weeks of culture. Furthermore, after about 6 weeks of culture, the capillaries disappeared from the OMS. After prolonged culture, tumour cell heterogeneity, the cellular composition, and the histology of the OMS still closely resembled the tumour in vivo. It is suggested that OMS provide a good long-term culture model for the study of gliomas.

Adult↗
PubMed1995

Cytolytic effects of autologous lymphokine-activated killer cells on organotypic multicellular spheroids of gliomas in vitro.

Knowledge about lymphokine-activated killer (LAK) cell infiltration and LAK cell cytotoxicity is essential to improve the effectiveness of LAK cell therapy against gliomas. In the present study, organotypic multicellular spheroids (OMS) of glioma tissue were used as a culture model to study the effects of LAK cells on gliomas. Compared to tumour cell lines and spheroids derived from tumour cell lines, OMS have several advantages with respect to preservation of tumour cell heterogeneity and the maintenance of the tumour architecture, e.g. capillaries and extracellular matrix. Four glioma specimens, obtained at surgery, were cultured directly on agarose to form OMS, which were then co-cultured with either autologous LAK cells or autologous non-activated peripheral blood lymphocytes (PBLs). After various time periods of co-cultivation, the OMS were fixed and examined both histologically and immunocytochemically. The present results showed that LAK cells infiltrated the OMS completely within 24 h of co-cultivation and severe cellular damage was observed, whereas PBLs infiltrated the OMS poorly and there was only marginal cellular damage. The present study indicates that OMS of gliomas provide an experimental model to investigate the infiltration and cytotoxicity of LAK cells on glioma tissue in vitro.

Adult↗
PubMed1994

Amplification of the anonymous marker D17S67 in malignant astrocytomas.

Loss of heterozygosity (LOH) for chromosome arms 9p, 10p, 10q, and 17p and amplification of the epidermal growth factor receptor (EGFR) gene have been identified as frequent genetic changes in malignant astrocytomas. We have found amplification of the anonymous marker D17S67 on chromosome arm 17p in 10% (3 of 30 cases) of astrocytomas of the highest malignancy grade. The tumors with D17S67 amplification displayed other genetic changes on chromosome 17, including additional amplifications and deletions. All three patients with D17S67 amplification developed severe brain edema and died within 1 month after operation.

Aged↗
PubMed1994

Allele loss on chromosomes 10 and 17p and epidermal growth factor receptor gene amplification in human malignant astrocytoma related to prognosis.

Patients with high-grade astrocytomas have a poor prognosis. However, considerable variation exists within this group of patients with respect to post-operative survival. In order to determine whether genetic alterations might be of help in subdividing this group, we used allele loss on chromosomes 10 and 17p and epidermal growth factor receptor (EGFR) gene amplification in the tumours as genetic parameters and determined their prognostic value. A series of 47 malignant (grade III and grade IV) tumours were genetically characterised, and four types of tumours were found. Type 1 tumours had loss of heterozygosity on chromosome arm 17p (LOH 17p) as the sole genetic alteration. Patients with this type of tumour were relatively young (mean age 39 years) and had a median survival period of 17 months. Type 2 tumours displayed only allele loss on chromosome 10 (LOH 10), type 3 tumours had LOH 10 + LOH 17p and type 4 tumours contained LOH 10 + EGFR gene amplification. Patients with types 2, 3 and 4 tumours were older (mean ages 59, 65 and 54 years respectively) and had a shorter survival (median duration 6, 3 and 2 months respectively) than type 1 patients. Multivariate analysis indicated that the genetic subdivision was a significant prognostic variable. In this study, age proved to be of minor importance with regard to survival. Our study revealed a predominance of frontally located tumours in patients with type 1 tumours, i.e. with LOH 17p only.

Adult↗
PubMed1994

Genetic versus histological grading in stereotactic biopsies.

With diagnostic stereotactic biopsies in astrocytoma sometimes false results will be encountered. A false positive result is defined as a histological diagnosis which is afterwards proved to be untrue. Although this happens very seldom (1-2%) in characterizing the tumor type (e.g. astrocytoma versus lymphoma), there is a probability of undergrading in malignant astrocytomas due to regional heterogeneity in malignancy grade. The issue of 'sampling error' has been addressed by many authors. It was shown that undergrading in astrocytomas is a likely event in fibrillar astrocytomas that often show geographically distinct areas of well and poorly differentiated elements. In a recent study on histological grading versus genetic characterization of heterogeneous astrocytomas we published that low-grade areas within high-grade malignant astrocytomas have already the genetic features of high-grade malignancy. Therefore, particularly in stereotactic biopsies which contain relatively small samples of tumor tissue, this fact is of paramount importance. Undergrading may lead to withholding radiotherapy from the patient and to falsification of long-term survival results in series of so-called low-grade astrocytomas. We like to stress the importance of taking biopsies from different parts of the tumor, especially from parts with different density enhancement to contrast with CT scanning, and will discuss the technical advancements made in the genetic grading of astrocytomas. Particularly loss of heterozygosity of chromosome 10 and eventually amplification of the EGFR indicate a high grade of malignancy.

Adult↗
PubMed1993

Endothelial cell marker PAL-E reactivity in brain tumor, developing brain, and brain disease.

BACKGROUND: The endothelial cell marker PAL-E is not reactive to vessels in the normal brain. The present study concerns the PAL-E reactivity in brain tumors in contrast to normal brain and nonneoplastic brain disease. METHODS: A total of 122 specimens were examined: brain tumors (n = 94), nonneoplastic brain disease (n = 19), normal brain (n = 8), and fetal brain (n = 1). Standard immunohistochemical procedures using a panel of endothelial cell markers were applied to detect vessels reactive to PAL-E. RESULTS: PAL-E reactivity to endothelial cells was found in all cases of glioblastoma multiforme, in 75% of the cases of anaplastic astrocytoma, and in 46% of the cases of astrocytoma. Furthermore, PAL-E reactivity was present in diseases with a developmental etiology, such as primitive tumors and congenital vascular malformations. The developing human brain (6-weeks' gestation age) and special sites of the mature brain, sites without blood-brain barrier, showed a strong reactivity, which indicates a relation with the status of blood-brain barrier development. CONCLUSIONS: PAL-E is the only marker out of a panel of endothelial cell markers that shows no reactivity to endothelial cells in the normal brain with an intact blood-brain barrier. In primary and metastatic brain tumors, PAL-E is reactive to endothelial cells, except for 25% of anaplastic astrocytoma and 54% of astrocytoma. PAL-E reactivity in brain tumors most likely is related to angiogenesis and to blood-tumor barrier properties not present in the normal blood-brain barrier.

Antibodies, Monoclonal↗
PubMed1992

Molecular characterization of areas with low grade tumor or satellitosis in human malignant astrocytomas.

Malignant astrocytomas often display histopathological heterogeneity. In the present study, we have molecularly characterized different areas within 4 such tumors to determine whether the tissue heterogeneity can be explained by differences in DNA constitution. Two tumors contained low grade areas, and the other 2 had areas with satellitosis. The tumors were examined for loss of heterozygosity with markers from chromosomes 9p, 10, and 17p and for amplification of the epidermal growth factor receptor gene. In each case, the high grade portion of the tumor displayed at least one of these structural alterations. However, identical alterations were found in the associated low grade or satellitosis areas of each tumor. Our data suggest that: (a) genetic alterations associated with tumor progression already occur in histopathologically low grade areas of high grade astrocytoma; (b) satellitosis associated with a high grade astrocytoma has to be considered as part of that tumor; and (c) tissue heterogeneity within a high grade astrocytoma is not a consequence of differences in DNA constitution at the loci that were examined.

Astrocytoma↗